Preparation Of Carboxymethyl Chitosan Based Composite Materials And Their Application In The Repair Of Bone Defect

Carboxymethyl chitosan(CMCS)is a derivatization of chitosan,which produced by the carboxymethyl modification of chitosan.CMCS is a soluble compound in water,which has good biocompatibility,biodegradability and pH sensitive properties.Furthermore,CMCS is able to chelate Ca2+ and has osteogenic activity and other characteristics,which has been widely used in the field of tissue repair.In recent years,New bone defect repair material is one of the popular research topics.This paper synthesized several kinds of composite materials based on CMCS and studied the feasibility of the related materials used in bone defect repair.The main research contents are as follows:First of all,in terms of the good water solubility of CMCS and easy to making films,the CMCS and silk fibroin(SF)composite membranes were prepared and crosslinked by genipin with good biocompatibility.The results showed that the crystal structure of the SF of the composite material transformed into phase Silk II from phase Silk I.The composite films showed a good hydrophilicity,which beneficial to the adhesion and proliferation of mouse bone marrow mesenchymal stem cells.Secondly,in views of the characteristics that CMCS can chelate Ca2+,CMCS used as regulation agent in the hydrothermal reaction,which precipitating HA and formed CACS/HA composite material.The effect of CMCS concentration and reaction temperature on the morphology of HA particles was discussed as well.The results showed that the structure of HA nanoparticle was changed from rod to sheet structure with the increasing CMCS concentration,and the effect of temperature on the morphology of the particles was negligible.The results of thermal analysis showed that when the concentration of CMCS in the reaction solution was 0.1%,0.2%,0.4%,0.6%,the content of CMCS in the composites was 15%,20%,25%,25% respectively.The composite material has good hydrophilicity and biocompatibility.The cell experiment shows that the material is beneficial to the adhesion and proliferation of bone marrow mesenchymal stem cells.In order to simulate the nanofiber morphology of extracellular matrix,CMCS nanofiber scaffold was prepared by electrospinning technique.Based on the good water solubility of CMCS,electrospinning can avoid the potential biological safety problems by using water as solvent.By adding polyoxyethylene(PEO),surface tension of the solution can be reduced effectively,and a detailed study of the influence of spinning voltage,molecular weight of PEO on nanofiber morphology has been done,the results showed nanofiber could been produced with good morphology when the molecular weight of PEO was 1 million with the voltage of 25 kV.CMCS/ calcium phosphate(CaP)composite nanofibers were prepared by biomimetic mineralization in 5 times simulated body fluid(5×SBF)for further improvement of the osteogenic activity of the nanofibers.The results showed that the nanofibers were favorable for cell adhesion and proliferation for both before and after mineralization,and the nanofibers after mineralization could promote the expression of alkaline phosphatase.RT-PCR showed that the nanofiber after mineralization can better promote gene expression of osteogenic markers(ALP,Runx-2,the main OCN and COL)and was more conducive to bone marrow mesenchymal stem cells to differentiate into osteoblasts compared with the nanofiber before mineralization.The SD rat model of calvarial defects was used to evaluate the bone repairing ability of nanofibers for both before and after mineralization,At 4 weeks after surgery,micro-CT,hematoxylin eosin staining(HE staining)and Masson trichrome stain(masson's trichrome)showed that the new bone formation started at the middle of the defect for mineralized nanofiber group and the new bone mainly growth from the edge of defect site for blank group and unmineralized nanofiber group.After 12 weeks,bone defect site fully covered with new bone in the mineralized nanofiber group and the bone defect was barely no healed in the blank group and the unmineralized nanofiber group.Finally,one of the most important problem need to be solved for applying CMCS as scaffolds for bone tissue engineering is that the CMCS is hydrophilic.This paper firstly used the reactive electrospinning technology,through the Schiff's base reaction between aldehyde of oxidized alginate(ADA)and the amino of CMCS,CMCS was crosslinked by ADA during electrospinning.This technology is expected to avoid the biological toxicity of glutaraldehyde.This paper discussed the influence of rheological behavior of the mixed solution?PEO molecules and the voltage on the morphology of nanofiber,the results show that when the molecular weight of PEO was 1 million,the voltage is 25 kV,the CMCS-ADA-PEO composite nanofiber with nice morphology could be acquired,through the method of soaking deionized water can effectively remove PEO from the mixture and get the CMCS-ADA nanofiber.After applying acid solution,alkali solution and PBS neutral solution immersion test,the results proved that the composite nanofiber scaffold has good resistance to water solubility.Cell experiment results showed that the introduction of ADA reducing the hydrophilic effect of the nanofiber.In the meantime,compare to the pure CMCS nanofibers and the CMCS nanofiber which crosslinked by glutaraldehyde,CMCS-ADA nanofibers are more conducive for cell adhesion and proliferation and differentiate into osteoblasts.